Molecular Basis of the Suppression of DNA
Replication Initiation by Cdc6
著者
Lena Rani Kundu
学位授与機関
Tohoku University
ಞ⒬ㄵᩝ
Molecular Basis of the Suppression of
DNA Replication Initiation by Cdc6
(Cdc6 ࡞ࡻࡾ DNA 々⿿㛜╂ᢒโࡡฦᏄᶭᵋ)
᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁ ༡ ⒬ㄚ⛤๑
⏍ⷾᏕᑍᨯ 㐿ఎᏄⷾᏕ ฦ㔕
Abstract
Given the immense size and complexity of eukaryotic genomes, duplication of chromosomal DNA presents a significant challenge for the cell. Therefore, initiation of DNA replication must be tightly regulated to ensure stable maintenance of the genome. This is achieved at first by the strict sequential binding of ORC, Cdc6, Cdt1 and Mcm2-7 forming the pre-replicative complex (pre-RC) and thereby licensing the origin. As each origin initiates replication, Mcm2-7 are displaced from it, moving along with or ahead of the replication fork as a DNA helicase. The majority of work in recent years has focused on identifying the relevant players involved in forming and activating pre-RCs, determining the order in which they associate with origins, and characterizing their cell cycle regulation. However, little is known about the regulation of these events in initiating DNA replication. Our understanding of the mechanism of pre-RC assembly, significance of each step, pre-RC structure and how this structure prepares the origin DNA for initiation remains limited. In this study, I focused on one of the licensing factors, Cdc6, in order to gain further insight into the significance of its regulation during the cell cycle. Studies in different systems suggested that despite functional similarities, the activity of Cdc6 seems to be differently regulated in different systems. Whatever may be the mechanism of regulating Cdc6 activity, all these observations suggested the need of controlling Cdc6 during DNA replication and my goal was to uncover the significance of this regulation in Xenopus egg extracts.
This research made use of the suitability of cell-free extracts of Xenopus eggs to analyze the precise and dynamic regulation in the process of origin activation. This study finds that Cdc6 is a key regulating factor even after licensing, influencing on the activation of Mcm2-7 by controlling the phosphorylation of one of its subunits, Mcm4. The molecular analysis of this finding is the main topic of Part 1. In Part 2 and 3, I made
use of fragmented and mutants of Cdc6 to dissect the phenomenon in deep and came up with another interesting result that Cdc6 likely has an effect on histone chaperone, FACT.
Previous studies showed that although Cdc6 is essential for DNA replication, Cdc6 becomes dispensable for the initiation of DNA replication once licensing has completed and its abundance is tightly regulated in the cell cycle. Therefore, it has been presumed that an excess of Cdc6 would result in overreplication or rereplication given the fact that Cdc6 is a positive regulator of the cell cycle. To our surprise, this study shows that external addition of Cdc6 inhibited DNA replication rather than inducing rereplication in Xenopus egg extracts when its concentration was about five times higher than the endogenous level. The inhibition by Cdc6 occurred at a step after pre-RC formation and before Cdk2-dependent steps as the addition of Cdc6 hardly influenced functional pre-RC formation, the chromatin binding of Cdk2, Cdk2-dependent origin firing or elongation of nascent DNA. The treatment, however, strongly suppressed phosphorylation of Cdc7 and Cdc7-dependent hyperphosphorylation of Mcm4 on chromatin. This inhibition was highly specific to Mcm4 as no inhibition of phosphorylation was observed for Mcm2 under these conditions. As a result, subsequent Cdk2-dependent chromatin loading of GINS complex and Cdc45 was blocked, suggesting that DNA replication was arrested after the loading of Cdc7/Drf1 and before the hyperphosphorylation of Mcm4. Therefore, I speculate that Cdc6, when present in excess, prevents the initiation of DNA replication by modulating the activity of Cdc7 to phosphorylate Mcm4 on chromatin, thereby blocking subsequent steps.
The interesting finding of the relationship between Cdc6 and FACT is now under progress. Until now, I found that Cdc6 destabilized FACT on chromatin, which probably occurred after origin licensing, and N-terminal Cdc6 was sufficient to cause
this destabilization. The relationship between Cdc6, Mcm4 and FACT will hopefully enrich our present understanding of the molecular mechanism of DNA replication initiation and Cdc6 function. Further analyses using various fragmented and mutant versions of Cdc6 will help us investigate this relationship in future and I am now on the way to it.
The main achievement of this study is the discovery of a novel regulatory mechanism of the initiation of DNA replication signifying the importance of a strict regulation of Cdc6 function during the course of DNA replication. The challenge in the future will be to decipher how Cdc6 functions together with other factors for replication initiation to turn the licensed origin into a replicating one.
ಞ⒬ㄵᩝཾ㢄Ⓠ⾪හᐖこ᪠
夢⫴ᬊ姥┘Ⓩ䚽 ┷ᰶ⏍∸套奛䚮DNA 々⿿夸ୌᅂ奚⣵⬂࿔套䛐䛊䛬ୌᗐ䛦䛗⾔䜕奸䜑䛥䜇奚โ ᚒ〓ᵋ奔䛝䛬DNA 々⿿姓䜨妥䝷妣〓ᵋ夸Ꮛᅹ䛟䜑夕奪䛠䚮ORC 夸妙䝱䝢䝅䝷套⤎ ྙ䛟䜑䛙奔套奴䜐々⿿㛜ጙⅤ夸Ử奪䜐䚮ぽ套䚮ORC ౪ᏋⓏ套 Cdc6䚮Cdt1 ཀྵ䛹 Mcm2-7 々ྙమ夸㡨ぽᑙ̶䛛奸䚮姓䜨妥䝷妣⇒ᚺ夸Ᏸ䛟䜑夕䛙奚⇒ᚺ奚ᚃ䚮 Cdc7 ཀྵ䛹 Cdk2 奚䝃䝷妻妙㈹委䝷㓗Ὡᛮ套౪Ꮛ䛝䛬ᵕ䚱䛰䝃䝷妻妙㈹夸〓⬗䛝䚮 DNA 々⿿夸㛜ጙ䛛奸䜑ᵕᏄ夸┉䛕奚◂✪ᠺᯕ套奴奏䛬リ⣵套⌦ゆ䛛奸奐奐央䜑夕奔 䛕套 Cdc7 奛姓䜨妥䝷妣ᅄᏄ奓央䜑 Mcm2-7 々ྙమ奚々ᩐ奚䜹䝚姐䝏䝇妲䜘委䝷㓗 䛝䚮䛙奸套奴奏䛬DNA 々⿿䜘姓䜨妥䝷妣௧ᚃ奚ミ㝭套㐅䜇䜑䛥䜇套㔔こ䛰ᙲ 䜘ᢰ奏䛬䛊䜑䛙奔夸♟䛛奸䛬䛊䜑夕௧୕奚奴夲套䚮⌟ᅹ奪奓套䚮姓䜨妥䝷妣套㛭䛟 䜑䝃䝷妻妙㈹奚Ὡᛮ䜊ິឺ夸 DNA 々⿿奚㐛⛤奓⣵夷䛕โᚒ䛛奸䜑ᵕᏄ夸᪺䜏夷套 䛛奸䛬䛓䛥夸䚮奉奚โᚒ奚ណ⩇䜊โᚒ奚◒⥚夸DNA 々⿿套ཀྵ奨䛟ᙫ㡢套奐䛊䛬奛 Ᏸධ套⌦ゆ䛛奸䛬䛊䜑奔奛ゕ䛊㞬䛊夕奉䛙奓䚮ᮇ◂✪奓奛≁套 Cdc6 套Ἰ┘䛝䚮奉奚 Ὡᛮโᚒ奚ណ⩇䜘᥀䜑䛥䜇䚮Xenopus ༵ฝᾦ↋⣵⬂♧㥺⣌䜘⏕䛊䛬ゆᯊ䜘⾔奏 䛥夕 夢⤎ᯕ姥⩻ᐳ䚽 奪䛠䚮┒⭘Ⳟ奓Ⓠ⌟䛛奇䛥⤄ᥦ䛎 Cdc6䟺rCdc6䟻䜘༵ฝᾦ套㐛㔖῟ຊ䛟䜑䛙奔 奓Cdc6 奚Ὡᛮโᚒ奚␏ᖏ䜘ᶅೊ䛟䜑䛙奔夸奓䛓䜑奔⩻䛎䚮奉奚㝷奚 DNA 々⿿㛭㏻ 奚〓⬗奚ን套奐䛊䛬ㄢ奥䛥夕Cdc6 奛 DNA 々⿿套ᚪ㡪奓央䜑䛥䜇䚮Cdc6 夸㐛 套Ꮛᅹ䛝䛥⑺ྙ套奛 DNA 々⿿夸ಀ㐅䛛奸䜑奔⩻䛎䜏奸䛥夸䚮套⇒䛝䛬䚮DNA 々⿿奚㢟ⴥ䛰ᢒโ夸びᐳ䛛奸䛥夕Cdc6 奛ᮅᏰ奚 DNA 々⿿䜘វ▩䛝⣵⬂࿔䜘 ೳイ䛟䜑䝅䜫䝇妙䝡䜨䝷妲〓ᵋ套㛭䜕䜑䛙奔夸▩䜏奸䛬䛊䜑䛥䜇䚮䛙奚䝅䜫䝇妙䝡䜨䝷妲 〓ᵋ奚㜴ᐐ奓央䜑caffeine 䜘῟ຊ䛝䛥᮪௲ୖ奓 rCdc6 ῟ຊ奚℃ᯕ套奐䛊䛬᳠ゞ䛝 䛥夸䚮䛙䛙奓ず䜏奸䜑DNA 々⿿奚ᢒโ奛䝅䜫䝇妙䝡䜨䝷妲〓ᵋ䜘䛟䜑奮奚奓奛䛰䛊 䛙奔䜘♟ြ䛟䜑⤎ᯕ夸ᚋ䜏奸䛥夕䛛䜏套䚮DNA 々⿿㛜ጙ⇒ᚺ䚮央䜑䛊奛䚮奉奚ᚃ奚᩺ ⏍㙈ఘ㛏⇒ᚺ套ᑊ䛟䜑℃ᯕ套奐䛊䛬᳠ゞ䛝䛥夕䛙奚䛥䜇套䚮Cdk2 㜴ᐐ䝃䝷妻妙㈹奓 央䜑p21 套奴奏䛬 DNA 々⿿㛜ጙ⇒ᚺ䜘ᢒโ䛝䛥᮪௲ୖ奪䛥奛 DNA 䝡委始姓姦䝀㜴 ᐐ䜦䝙妍䝋妍妝委䝷奓᩺⏍㙈ఘ㛏ミ㝭䜘ೳイ䛛奇䛥᮪௲ୖ奴䜐⣵⬂ᰶ䜘༟㞫䛝䚮 rCdc6 䜘῟ຊ䛝䛥༵ฝᾦ୯奓 DNA 々⿿䜘 㛜䛛奇䛥夕奉奚⤎ᯕ䚮rCdc6 奚῟ຊ套奴䜑 DNA 々⿿奚ᢒโ奛䚮Cdk2 套奴䜑 DNA 々⿿㛜ጙὩᛮ௧๑奚㐛⛤䛾奚℃ᯕ 奓央䜑䛙奔夸♟䛛奸䛥夕
ぽ套䚮rCdc6 䜘῟ຊ䛝䛥༵ฝᾦ奴䜐妙䝱䝢䝅䝷⏤ฦ䜘༟㞫䛝䚮姓䜨妥䝷妣㜴ᐐ䝃䝷
妻妙㈹ geminin 䜘ྱ奬༵ฝᾦ୯奓 DNA 々⿿䜘 㛜䛛奇䛥奔䛙䜓䚮DNA 々⿿Ὡᛮ
夸☔ヾ䛛奸䛥䛙奔夷䜏䚮rCdc6 奚῟ຊ套奴䜑༵ฝᾦ୯奚姓䜨妥䝷妣Ὡᛮ䛾奚ᙫ 㡢奛䛰䛊䛙奔夸♟ြ䛛奸䛥夕奉䛙奓䚮姓䜨妥䝷妣௧ᚃ䚮DNA 々⿿㛜ጙ௧๑奚ミ㝭 套Ἰ┘䛝䛬リ⣵䛰᳠ゞ䜘⾔奏䛥夕奉奚⤎ᯕ䚮DNA 々⿿奚㐛⛤套౪Ꮛ䛟䜑奮奚奔⩻䛎 䜏奸䜑Mcm4 奔 Cdc7 奚 SDS-PAGE ୕奓奚᪾ິᗐ奚ን夸 rCdc6 奚῟ຊ套奴䜐㢟 ⴥ套ᢒโ䛛奸䛬䛊䛥夕䛙奚᪾ິᗐ奚ን奛⬲委䝷㓗㓕⣪ฌ⌦套奴奏䛬奮ゆ㝎䛛奸 䜑䛙奔奴䜐䚮rCdc6 奚῟ຊ夸 Cdc7 套奴䜑 Mcm4 奚委䝷㓗⇒ᚺ䜘ᢒโ䛝䛬䛊䜑奮奚奔 ⩻䛎䜏奸䛥夕奪䛥䚮DNA 々⿿套ᚪ㡪奓央䜐 Cdc7 夸ᐞ䛟䜑ミ㝭௧㜾奓妙䝱䝢䝅䝷套 ⤎ྙ䛟䜑GINS 々ྙమ䚮Cdc45䚮ୌᮇ㙈 DNA ⤎ྙ䝃䝷妻妙㈹ RPA 奚妙䝱䝢䝅䝷⤎ྙ 奮ᢒโ䛛奸䛬䛊䛥䛙奔夷䜏䚮Cdc6 奚〓⬗夸ஸ㐅䛟䜑䛙奔套奴奏䛬 Cdc7 夸ⅴฦ套〓⬗ 奓䛓䛰䛕䛰䜐䚮䛙奸套奴奏䛬奉奸௧㜾奚ミ㝭夸㐅⾔奓䛓䛰䛕䛰奏䛬 DNA 々⿿夸ᢒโ䛛 奸䜑奮奚奔᥆ῼ䛛奸䛥夕 Cdc6 奚〓⬗奚ஸ㐅套奴䜐 Cdc7 奚 kinase Ὡᛮ夸㜴ᐐ䛛奸䜑ྊ⬗ᛮ夸⩻䛎䜏奸䛥䛥 䜇䚮⢥⿿⤄ᥦ䛎䝃䝷妻妙㈹䜘⏕䛊䛥Cdc7 套奴䜑 Mcm2467 々ྙమ奚委䝷㓗⇒ᚺ套 ᑊ䛟䜑 rCdc6 奚℃ᯕ套奐䛊䛬᳠ゞ䛝䛥夕䛝夷䛝䛰夸䜏䚮䛙奚♧㥺⣌奓奛 Cdc7 奚委䝷 㓗Ὡᛮ䜘rCdc6 夸ᢒโ䛟䜑䛙奔䛰夷奏䛥夕奪䛥䚮Cdc7 奚奮夲ୌ奐奚ᵾⓏ奔⩻䛎䜏奸 䛬䛊䜑Mcm2 奚委䝷㓗套奐䛊䛬奮༵ฝᾦ䜘⏕䛊䛬᳠ゞ䛝䛥夸䚮rCdc6 䜘῟ຊ䛝䛥 ᮪௲ୖ奓奛Mcm2 奚委䝷㓗≟ឺ䜘♟䛟 SDS-PAGE ୕奓奚᪾ິᗐ奚㐼⛛奛ᢒโ䛛 奸䛰夷奏䛥夕௧୕奚⤎ᯕ夷䜏䚮Cdc6 奛 Cdc7 奚䝃䝷妻妙㈹委䝷㓗Ὡᛮ 䜘㜴ᐐ䛟䜑 奚奓奛䛰䛕䚮妙䝱䝢䝅䝷୕套⛸䝃䝷妻妙㈹夸㞗ྙ䛝䛥᮪௲ୖ奓䚮Mcm4 奚委䝷㓗 ᵾⓏ㒂న䛾奚 Cdc7 奚㎾䜘ᢒโ䛝䚮䛙奸套奴奏䛬䚮Mcm4 套ᑊ䛟䜑委䝷㓗䜘≁␏ Ⓩ套ᢒโ䛝䛬䛊䜑䛙奔夸♟ြ䛛奸䛥夕 ᮇ◂✪套奴䜐ᚋ䜏奸䛥⤎ᯕ奛䚮DNA 々⿿㛜ጙ奚ㅎミ㝭套䛐䛗䜑 Cdc6 奚⥝ᐠ䛰Ὡ ᛮโᚒ奚ណ⩇䜘⌦ゆ䛟䜑୕奓㔔こ䛰▩ず奓央䜑䛦䛗奓䛰䛕䚮Cdc6 奚โᚒ套奴䜑᩺ぜ 奚DNA 々⿿㛜ጙㄢ⟿〓ᵋ夸Ꮛᅹ䛟䜑ྊ⬗ᛮ䜘ᥞ♟䛟䜑奮奚奓央䜑夕
Table of Contents
Acknowledgements
Part 1: Regulation of the initiation of DNA replication by Cdc6
1 Introduction ……….………..14
1.1 Cell cycle ………...14
1.2 DNA replication ………....16
1.2.1 Origin licensing ………..16
1.2.2 The initiation of DNA replication ………..17
1.3 Cdc6: the central character of this study ………..…….19
1.4 Xenopus egg extract cell-free system : a powerful tool for biochemical analyses ………..22
2 Purpose of this study .………24
3 DNA Replication under additive Cdc6 conditions………..26
3.1 Purification of recombinant Cdc6 ………..26
3.2 DNA replication is strongly inhibited in rCdc6supplemented extracts..…...27
3.3 Evaluation of rCdc6 activity……….…..28
4 Cdc6 and DNA replication checkpoint pathways ...………...30
4.1.1 Sensors ………...31
4.1.2 Transducers .………...32
4.1.3 Effectors ……….33
4.2 Signal propagation at the stalled fork ………34
4.3 Cdc6 inhibits DNA replication without activating ATM/ATR-dependent checkpoint pathways .……….35
4.3.1 Immunoblot analysis .………..35
4.3.2 Replication assays by the incorporation of radioactivity .………...36
5 Effect of addition of Cdc6 on the course of DNA replication .………...38
6 Effect of addition of Cdc6 on DNA synthesis .…………..………..39
6.1 DNA polymerases .……….39
6.2 Aphidicolin: a DNA polymerase inhibitor .………39
6.3 Strategy and assay designing ……….40
6.4 DNA synthesis was not inhibited by addditive Cdc6 ...……….41
7 Effect of additive Cdc6 on Cdk2-dependent steps ……….42
7.1 CDKs: the major regulator of the cell cycle………...42
7.2 p21: an inhibitor of Cdk2………...43
7.4 DNA replication is inhibited by additive Cdc6 at a step before
Cdk2-dependent pathways ...………..45
8 Influence of additive Cdc6 on origin licensing ..……….46
8.1 Introduction………....….46
8.1.1 ORC……….46
8.1.2 Cdt1 and geminin………....….46
8.1.3 Mcm complex (Minichromosome maintenance complex)………..47
8.2 Cdc6 does not afftect functional pre-RC formation………48
8.2.1 Immunoblot analysis………48
8.2.2 Additive Cdc6 did not hamper functional pre-RC formation…………..49
9 Influence of additive Cdc6 on phosphorylation state of subunits of Mcm2-7 complex...53
9.1 Post-translational modifications of Mcm2-7 subunits………...53
9.2 DDK (Cdc7/Dbf4 and Cdc7/Drf1 kinase complexes)………..….56
9.3 Cdc6 arrests DNA replication before Mcm4 phosphorylation …...………...57
9.4 Cdc6 inhibits hyperphosphorylation of Mcm4, but not Mcm2…………...58
Part 2: Analysis of fragmented and WA, WB mutants of Cdc6
1 Analysis of N-terminal Cdc6 fragment (N-Cdc6)………63
1.1 Introduction ……….63
1.2 Effect of N-Cdc6 on DNA replication ………64
1.3 N-Cdc6 inhibits DNA replication earlier than Cdk2-dependent pathways .…66 2 Analysis of WA, WB mutants of Cdc6 .……….68
2.1 Introduction ……….………68
2.2 Effect of WA and WB mutant of Cdc6 on DNA replication ………..69
2.3 WA- and WB-Cdc6 inhibited DNA replication at the step of origin licensing without the activation of ATM/ATR-dependent checkpoint pathways………..70
Part 3: Cdc6 and its effect on histone chaperone, FACT
1 Introduction……….722 Effect of Cdc6 on the chromatin binding of FACT………..74
2.1 Chromatin binding of FACT was limited in the presence of additive Cdc6 ...74
2.2 Cdc6 destabilized FACT on chromatin ..………75
3 Destabilization of FACT was not directly dependent onto Cdc7 activity……..77
Discussion………..….80
Materials and methods………..…89
Acknowledgements
Many people have directed and supported me to accomplish this research and, therefore, my debts are endless.
First, I would like to thank Professor Takemi Enomoto, my supervisor, for giving me the opportunity to explore such an interesting and promising subject. His kind-hearted guidance and encouragement have greatly helped me to accomplish this research.
I would like to express my heartful gratitute to Professor Shoichiro Kurata for critically reading this manuscript and encouraging discussions.
I would like to express my heartful gratitude to Assisstant Professor Shusuke Tada for his insightful comments and kind guidance throughout the course of this research. Without his supervision, this work would have been much less than it is.
I would like to sincerely thank Associate Professor Masayuki Seki and Assisstant Professor Akari Yoshimura for their insightful comments and helpful suggestions.
I would like to express my special thanks to Yuji Kumata for showing me the first steps in this scientific world. His careful supervision and meaningful suggestions helped me mold the philosophy with which I approached questions of research. I am immensely grateful for his support and proud to have him as my direct instructor.
I would like to thank all the members of my laboratory for their interests and critical responses to my research activities. Special thanks to Eri Inoue and Mong Sing Lai whose unwavering love and support helped me follow my path and grow.
I would like to express my gratitude to the Japanese Ministy of Education, Culture, Sports and Technology for the financial support throughout the years. Without this support my stay in Japan would have been impossible.
Finally, I would like to thank my motherland, Bangladesh, for nurturing my growth in her loving womb. My eternal thanks to my family for their presence in my existence and for the love and support throughout my life. They believed in me even when I did not believe in myself. They taught me to have faith and always showed me the right path to follow. I am grateful to you all.
Part 1
Regulation of the initiation of DNA replication by Cdc6
1 Introduction
The discovery that DNA has a base-paired double-helical structure was a landmark in 20th century biology because it suggested an obvious mechanism for how genetic information is replicated and transmitted to daughter cells. Since then, the basic proteins involved in DNA replication, such as DNA polymerases and helicases, have been discovered and extensively studied. Now we are beginning to reveal the mechanisms by which DNA replication is initiated at particular positions on chromosomes, called origins, and to determine why replication occurs only during S phase and how over-replication of the genome is avoided. Although yeasts have been the frequent subjects of study in eukaryotic DNA replication, DNA replication in metazoans is not identical to that in yeasts and in fact has a number of unique characteristics. That is why different approaches from several viewpoints have been carried out using multiinformative biochemical tools. This chapter will introduce the basics of DNA replication, related mechanisms and experimental system I have used in this study.
1.1 Cell Cycle
The cell cycle is a critical regulator of the processes of cell proliferation and growth as well as of cell division after DNA damage. It governs the transition from quiescence
(G0) to cell proliferation, and through its checkpoints, ensures the fidelity of the genetic transcript. It is the mechanism by which cells reproduce, and is typically divided into four phases.
Figure 1-1-1. A schematic representation of the cell cycle.
The periods associated with DNA synthesis (S phase) and mitosis (M phase) are separated by gaps of varying length called G1 and G2 (gap 1 and gap 2). G1 and G2 are characterized by protein and RNA synthesis, but no DNA synthesis. S (synthesis) is the period of DNA synthesis. M (mitosis) is the period when the ell divides. G0 is a "resting" stage and may be omitted when cells are continuously cycling. The timing of the cell cycle and the relative lengths of the various stages depend on the specific type of cell and on the local conditions. At the completion of cell division, the daughter cells
may re-enter the cell cycle or may undergo terminal differentiation.
1.2 DNA Replication
1.2.1 Origin licensing
The eukaryotic genome is organised into multiple chromosomes, whose replication must be strictly controlled in order to ensure that the DNA is replicated once and only once in each cell cycle. DNA replication initiates from multiple origins, whose activation can be divided into two stages. In the first stage, pre-RCs are assembled at replication origins by the sequential binding of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 (the MCM/P1 proteins). This assembly takes place during late mitosis and G1, and results in the origin becoming "licensed" for DNA replication. “Licensing” mechanism ensures that DNA replication occurs once and only once in each cell cycle and was first proposed by Blow and Laskey in 1988. The basic concepts of origin licensing are listed below:
(1) Licensing factors are essential for DNA replication.
(2) Licensing factors associate with chromatin before S phase.
(3) Licensing factors are degraged or become inactivated as DNA replication proceeds.
(4) Licensing factors cannot cross nuclear membrane, therefore, cannot bind DNA until nuclear envelope breaks down in next M phase ensuring a single round of DNA replication in each cell cycle.
Figure 1-1-2 Steps of origin licensing in eukaryotes.
Therefore, origins licensed in G1 phase can only respond to signals for DNA replication in S phase. The following M phase enables licensing factors to activate origin again and prepares for the DNA replication in the next cell cycle. The factors directly involved in licensing are ORC, Cdc6, Cdt1, and Mcm2-7 complex.
1.2.2 The initiation of DNA replication
The second stage occurs during S phase and involves the activation of licensed origins by the action of two S phase-promoting kinases: S-phase promoting cyclin-dependent kinases (CDKs) and Cdc7/Dbf4 (DDK), leading to the loading of Cdc45 and the formation of a pair of replication forks.
In metazoans, Mcm10 and the protein kinase Cdc7/Dbf4 are the first factors to be loaded onto pre-RCs, and their loading is Mcm2̽7-dependent. Mcm10 and Cdc7 enable the loading of several additional factors such as GINS and Cdc45, whose binding is also dependent on Cdk2/Cyclin E. The binding of Cdc45 and GINS to pre-RC is interdependent and converts this structure into a pre-Initiation Complex (pre-IC).
Formation of the pre-IC is the last known event that occurs before origin unwinding, which is accompanied by chromatin loading of the single-stranded DNA-binding protein complex, RPA. Once the origin has been sufficiently unwound, DNA polymerase (pol) α loads and synthesizes an RNA primer, which it then extends to form a short DNA primer. The presence of a DNA primer allows loading of the polymerase clamp, PCNA, by the clamp-loader complex, RFC, followed by loading of pol δ.
Figure 1-1-3. Schematic representation of the steps to initiation of DNA replication in
eukaryotes. Phosphorylation of Mcm2-7 complex
DNA synthesis
Origin licensingCdc7
The final stage of DNA replication, elongation, involves the coordinated synthesis of nascent strands. Studies in yeast clearly show that, in addition to DNA polymerases, elongation also requires Mcm2-7, Cdc45, and GINS, all of which localize to replication forks. On the other hand, it is unknown whether Mcm2̽7, Cdc45, or GINS are also required for elongation in metazoans.
1.3 Cdc6: the central character of this study
Saccharomyces cerevisiae Cdc6 protein and its homolog in Schizosaccharomyces pombe, Cdc18, were identified almost three decades ago in temperature-sensitive cell-cycle mutants showing defects in the initiation of replication. In both yeasts, the protein is essential for the initiation of DNA replication and for loading the Mcm2-7 proteins onto chromatin. Cdc6 binding to mammalian and Xenopus chromatin in in vitro replication systems has also been shown to be a crucial early step in higher-eukaryotic DNA replication, and reduction in levels of Cdc6 is commonly associated with loss of proliferative capacity in human cells. Furthermore, Cdc6 inactivation seems to be an important event during programmed cell death to prevent a wounded cell from replicating and to facilitate its death.
1.3.1 Structure of Cdc6
Xenopus Cdc6 (XCdc6) is a 554 amino acid protein representing a band around 63 kDa by SDS-PAGE.
The important features of Cdc6 are listed below.
1. N-terminal domain (NTD) is regulatory and is not directly involved in pre-RC assembly.
2. The N-terminal region contains a putative nuclear localization signal.
3. Human Cdc6 1-192 amino acid region binds to Cdt1, but decreases Mcm2-7 loading. The critical elements lie between amino acids 125-165.
A.
B.
Figure 1-1-4. A. Secondary structure of Pyrobaculum aerophilum Cdc6 constituting of 3 major
domains: domain I, II and III. Domain I containing ATPase motif and domain III containing winged-helix domain (WHD) required for DNA binding are connected with domain II. B. Schematic representation of the structural features of Cdc6.
4. XCdc6 residues 143-164 form anȘ-helix that stabilizes the interface of the two domains that comprise the nucleotide binding site.
5. Majority of the SP or TP motifs are phosphorylated by CDK.
Cdc6 is a good substrate for cyclin E-, A-, B-associated CDKs and Ser 54, 74, 106 are known to be phosphorylated by CDKs in human cells. Two of these lie in close proximity to conserved destruction boxes recognized by the anaphase promoting complex (APC/C). The first Ser 54 is next to an RXXL-type destruction box while the second (Ser 74) is near a KEN-type destruction box. The RXXL motif is found in all of the vertebrate Cdc6 sequences, while the KEN box is present in all of the vertebrate Cdc6 sequences except XCdc6a, an isoform of Cdc6, specific for early Xenopus embryos. Xenopus embryos also lack Cdh1, which is involved in recognizing the KEN box. CDK phosphorylation-dependent masking of destruction boxes in Cdc6 directly prevents APC/C-dependent proteolysis.
6. The N-terminal domain shows significant homology to the ATPases associated with various cellular activities (AAA+) family of ATPases and encompasses a characteristic purine nucleotide binding site composed of Walker A and Walker B motifs. An intact Walker A motif is necessary for efficient XCdc6 chromatin association, whereas an intact Walker B motif is critical for Mcm2-7 loading and XCdc6 dissociation from chromatin.
7. Cdc6 functions in the formation of pre-RCs at a step after ORC binding, and its recruitment requires ORC, but how does Cdc6 bind to chromatin? Recent structural data based on the archaebacteria Pyrobaculum aerophilum Cdc6 protein and sequence comparisons have identified a winged-helix domain in the C-terminal region of Cdc6 proteins. Winged-helix domains are present in a number of
transcription factors and are responsible for protein̽DNA interactions. Although evidence for DNA binding of Cdc6 by this domain is still lacking, it is attractive to speculate that Cdc6 might contact chromatin through the winged-helix motif, perhaps by protein-protein interactions with the ORC.
The essential function of Cdc6 is to load the Mcm2-7 complexes onto chromatin, and studies in different systems support the idea that this is dependent on Cdc6 binding and hydrolyzing ATP. Cdc6 is a member of the AAA+ family of proteins and, interestingly, is similar in sequence to the replication factor RF-C that loads PCNA onto DNA. Based on this observation, it has been suggested that Cdc6 might perform a function similar to that of polymerase clamp loaders in the ATP-dependent loading of Mcm2-7 complexes around DNA.
8. C-terminal two-thirds of Cdc6 can restore DNA replication to ~20% of wild-type XCdc6 activity.
9. Low concentrations of Cdc6 that are sufficient to replicate DNA efficiently can form functional hetero-oligomers. It has been shown previously that Xenopus and human Cdc6 forms complexes in solution and this kind of oligomerization is greatly stabilized in the presence of DNA and other DNA replication components.
1.4 Xenopus Egg Extract Cell-free System: a powerful tool for biochemical analyses
Cell-free extracts derived from the eggs of Xenopus laevis are a powerful experimental system to study genomic DNA replication in higher eukaryotes. DNA templates (either plasmid DNA or Xenopus sperm chromatin) added to these extracts are packaged into
This process occurs rapidly and efficiently, and can be extensively manipulated in order to elucidate underlying biochemical mechanisms.
In contrast to budding yeast, where replication initiates at defined origin sequences, replication in Xenopus egg extracts initiates at apparently random DNA sequences. Importantly, replication in egg extracts requires the initiation factors ORC, Cdc6, Mcm2-7, Cdk2, Cdc7, and Cdc45, and initiation in Xenopus embryos is similarly sequence independent. These observations argue that the mechanism of initiation in Xenopus egg extracts is physiological, and for convenience, the sites where replication initiates in this system are referred to as "origins."
This cell-free system has proven extremely useful for studying initiation and elongation of genomic DNA replication, the spatial organization and structural requirements for DNA replication, the relationship of S phase to other phases of cell cycle, and the regulatory system which limits DNA replication to once per cell cycle.
2 Purpose of this study
The field of DNA replication has made enormous progress during the last decade. Relevant players have been identified one after another, and the order in which they bind origins or how they are regulated during the cell cycle have been the main attractions of scientists. However, our understanding of the mechanism of pre-RC assembly, significance of each step, pre-RC structure and how this structure prepares the origin DNA for initiation remains limited. Cdc6 has been one of these well-cultured, yet ambiguous topics.
The fate of Cdc6 during cell cycle stages varies in different organisms. In yeasts, phosphorylation of Cdc6 (or its homologue Cdc18 in Schizosaccharomyces pombe) by Cdks targets it for ubiquitin-dependent proteolysis. Cdc6 is rapidly phosphorylated and degraded when yeasts enter S phase, but Cdc6 is comparatively stable in frog eggs and mammalian cells. In metazoans, chromatin-bound Cdc6 persists throughout S phase and G2 and is degraded during G1 through ubiquitination by APC/C. During S phase and G2, however, the majority of the soluble (non-chromatin bound) Cdc6 appears to be exported out of the nucleus in a Cdk-dependent manner. The majority of the data showing Cdc6 nuclear export in S phase were obtained by over-expression of Cdc6. Surprisingly, a recent study focusing entirely on endogenous Cdc6 showed that even non-chromatin bound Cdc6 may remain nuclear throughout S phase. Despite this persistence of Cdc6 on chromatin or in nuclei later in the cell cycle, it has been shown in Xenopus that once DNA has been licensed, efficient DNA replication no longer requires the presence of Cdc6. In human, Cdc6 mutants that lack Cdk phosphorylation sites block initiation of DNA replication in some studies (Jiang et al., 1999, Herbig et al.,
2000), but not in others (Petersen et al., 1999; Pelizon et al., 2000). Moreover, although such Cdc6 mutants accumulate in the nuclei assembled in Xenopus egg extracts where they support initiation of DNA replication, they do not induce endoreduplication (Petersen et al., 1999; Pelizon et al., 2000).
The importance of regulating Cdc6 function is evident in fission yeast, where overexpression of the homologous protein, Cdc18, leads to multiple rounds of DNA replication without an intervening mitosis. However, misregulation of Cdc6 function is not sufficient to induce over-replication in S. cerevisiae and higher eukaryotes as over-replication is not observed even when an unphosphorylatable (and hence undegradable) mutant of Cdc6 protein is overexpressed. A recent study in S. cerevisiae has shown that replication is controlled by at least three overlapping mechanisms, which involve phosphorylation of the ORC, down-regulation of Cdc6 activity and phosphorylation of the Mcm2-7 complex, which results in removal of the Mcm2-7 complexes from chromatin and their nuclear exclusion. Similarly, redundant mechanisms are likely to exist in higher eukaryotes, and the multiple levels of regulation of pre-RCs could explain why disrupting Cdc6 regulation in humans and Xenopus is not sufficient to induce over-replication.
All the above studies in different systems suggested that despite functional similarities, the activity of Cdc6 seems to be differently regulated in different systems. Whatever may be the mechanism of regulating Cdc6 activity, all these observations suggested the need of controlling Cdc6 during DNA replication and a goal of the present research was to uncover the significance of this regulation in Xenopus egg extracts.
3 DNA replication under additive Cdc6 conditions
3.1 Purification of recombinant Cdc6
In order to gain insight into Cdc6 function during DNA replication, GST-fused recombinant Cdc6 (rCdc6) was produced in E. coli. Purified protein fraction was electrophoresed in SDS-10% polyacrylamide gel and stained with Coomassie Brilliant Blue. rCdc6 was identified by Coomassie Staining (Figure 1-3-1A) and was reconfirmed by Western Blotting (Figure 1-3-1B).
Figure 1-3-1. Purification of recombinant GST-Cdc6 (rCdc6). A. Coomassie Brilliant Blue
staining of rCdc6 fractions at different stages of purification. B. rCdc6 fractions at different stages of purification were subjected to SDS-PAGE and immunoblotted for Cdc6.
3.2 DNA replication is strongly inhibited in rCdc6-supplemented extracts
In order to learn the consequences of high levels of Cdc6 during chromosome replication in Xenopus egg extracts, DNA synthesis was measured in interphase extracts supplemented with various concentrations of rCdc6.
Figure 1-3-2. DNA synthesis was inhibited in rCdc6-supplemented extracts. (A) DNA
synthesis in Xenopus egg extracts supplemented with various concentrations of GST-Cdc6. DNA synthesis was assayed after 90 min of incubation by incorporation of radioactivity into nascent DNA from [a-32P]dATP. (B) Time course of DNA replication in extracts supplemented with buffer or GST-Cdc6 (700 nM). Amount of DNA synthesized at 0, 30, 45, 60, 90 min was assayed by incorporation of radioactivity into nascent DNA from [a-32P]dATP.
To our surprise, as the concentration of Cdc6 increased, the amount of the newly synthesized DNA decreased drastically between 500 nM and 700 nM concentration and DNA replication was completely inhibited at as low as 800 nM (Figure 1-3-2A). For a
double check, we examined the time course of DNA replication in extracts supplemented with or without GST-Cdc6 and a complete inhibition was confirmed in GST-Cdc6-supplemented extracts (Figure 1-3-2B). The inhibition was observed typically around this concentration, which varied slightly depending on the preparation but the overall tendency remained the same. Throughout this study, I used 700 nM of rCdc6 unless mentioned otherwise.
3.3 Evaluation of rCdc6 activity
To exclude the possibility that the inhibition was caused by non-protein component in the preparation, rCdc6 fraction was boiled and added to Xenopus egg extracts to compare with non-treated fraction. While non-treated rCdc6 caused complete inhibition of DNA replication, boiled fraction or the same concentration of BSA did not cause significant suppression of DNA synthesis (Figure 1-3-3).
Figure 1-3-3. Evaluation of rCdc6 activity. Egg extracts were supplemented with buffer,
rCdc6, denatured rCdc6 fraction and BSA. DNA synthesis was measured after 90 min of incubation.
To confirm that the rCdc6 preparation was functional, I assayed GST-Cdc6 for its ability to replace the native Cdc6 after immunodepletion of the egg extracts (data not shown). The result confirmed that depletion of the egg extract with antibodies raised against Cdc6 abolished the ability to support replication of sperm chromatin and the replication capacity was restored by addition of GST-Cdc6 indicating the fraction was fully functional.
In addition, we used N-terminal His-FLAG-tagged Cdc6 to confirm that this inhibition was not caused by GST tag fused to Cdc6 (data not shown). Finally, I repeated the experiment using Cdc6 fraction partially purified from Xenopus egg extracts, which was also able to block DNA replication sufficiently. These data support the conclusion that Cdc6 blocks DNA replication at higher concentrations in Xenopus egg extracts.
4 Cdc6 and DNA replication checkpoint pathways
4.1 Checkpoint pathways
Studies with yeast, invertebrates, frogs, and mammals have revealed sets of proteins that play roles in the signaling pathways involved in responding to extrinsic and intrinsic damage during S phase. DNA damage checkpoints are biochemical pathways that delay or arrest cell cycle progression in response to DNA damage. Originally a checkpoint was defined as a specific point in the cell cycle when the integrity of DNA was examined (“checked”) before allowing progression through the cell cycle. Although the checkpoint pathways are operational during the entire cell cycle and hence may slow down the cell cycle at any point during the four phases, the most renowned checkpoint pathways are G1/S, intra-S, G2/M, and mitotic spindle checkpoints. The DNA damage checkpoint, like other signal transduction pathways, conceptually has three components: sensors, signal transducers and effectors (Figure 1-4-1).
Figure 1-4-1. Schematic representation of the basic steps of checkpoint pathways.
Replication stress/DNA damage
damage
Sensors
Transducers
Effectors
Slowing down or pause
in the cell cycle
4.1.1 Sensors
ATM
DNA double strand breaks (DSBs) can take place during unperturbed DNA replication as a consequence of stalled forks or oxidative stress. Moreover, DSBs are necessary in cellular processes such as meiosis. Ataxia telangiectasia-mutated protein (ATM) has a major role in sensing this particular type of damage and a complex of three proteins Mre11, Rad50 and Nbs1 (Xrs2 in yeast) termed the MRN complex that contributes to recruitment of active ATM to sites of DSBs. Mre11 has an exonuclease activity while Rad50 and Nbs1 stimulate Mre11 enzymatic activity. Nbs1 has also a BRCA1 C-terminal domain (BRCT) that is responsible for protein̽protein interactions between checkpoint-related molecules.
ATR/ATRIP
While hATM seems to play a key role in responding to DSBs after ionizing radiation (IR), hATR (Atm- and Rad3-related) is activated after a wider variety of insults including UV light, HU-dependent replication inhibition and DNA methylation by methyl methane sulfonate (MMS). ATR, the primary S phase checkpoint kinase, plays roles in both damage sensing and DNA replication. This is in contrast to ATM which may only sense damaged DNA. In fact, the broader spectrum of ATR activating signals correlates with the much higher lethality of ATR loss. ATR binds to ATRIP which works as a regulatory subunit. Deletion of ATRIP renders the cell effectively ATR-null. ATR can phosphorylate both ATRIP and RPA although the relevance of such phosphorylation is not known currently. The ATR/ATRIP complex associates with RPA-coated DNA independently of any checkpoint proteins, which suggests that this complex directly recognizes damaged DNA. ATR not only binds to damaged DNA but
has also been shown to interact with replicating DNA. To associate with unperturbed DNA replication forks, the ATR/ATRIP complex requires the previous loading of RPA on replicating ssDNA. This event does not require Pol α suggesting that ATR appearance in replicating forks takes place even before Pol α recruitment. The early association of ATR with replication forks is consistent with its newly identified role in the modulation of the timing of origin firing during unperturbed replication. Other ATR functions in the absence of stress could include scanning for changes in the speed and processivity of polymerases or in the extent of ssDNA accumulation at the fork. In all cases, full activation of ATR/ATRIP requires the independent loading of a second complex, Rad17/9-1-1, onto DNA.
4.1.2 Transducers
Chk1 & Chk2
In humans, there are two kinases, Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2 (Chk2), with a strictly signal transduction function in cell cycle regulation and checkpoint responses. These kinases were identified based on homology with yeast scChk1 and scRad53/spCds1, respectively. ATM or ATR phosphorylate these kinases as a direct consequence of checkpoint activation and these phosphorylated forms of Chk1 and Chk2 are good markers of ATM/ATR-dependent checkpoint pathways in biochemical experiments. Both Chk1 and Chk2 are S/T kinases with moderate substrate specificities. In mammalian cells, the double-strand break signal sensed by ATM is transduced by Chk2 and the UV-damage signal sensed by ATR is transduced by Chk1. However, there is some overlap between the functions of the two proteins.
4.1.3 Effectors
In humans, three phosphotyrosine phosphatases, Cdc25A, -B, and - C, dephosphorylate the Cdks that act on proteins directly involved in cell cycle transitions. Phosphorylation of these Cdc25 proteins by the checkpoint kinases creates binding sites for the 14-3-3 adaptor proteins, of which there are 8 isoforms. Phosphorylation inactivates the Cdc25 by excluding them from the nucleus, by causing proteolytic degradation, or both. Unphosphorylated Cdc25 proteins promote the G1/S transition by dephosphorylating Cdk2 and promote the G2/M transition by dephosphorylating Cdc2 phosphotyrosine.
(adapted and modified for clarity from Paulsen et al., 2007 and Sancar et al.,)
4.2 Signal propagation at the stalled fork
(1) During the initiation phase of DNA replication the pre-RC MCM complex and CDC45 load onto origins of replication. RPA binds to regions of single stranded DNA. ATR/ATRIP complexes are then loaded onto chromatin by direct binding to RPA. After Pol α primase loading, Rad17/9-1-1 complexes are recruited to the newly unwound fork. It is not clear if the ATR/ATRIP complexes are actively signaling at this stage.
(2) During the elongation phase of DNA replication the RFC/PCNA clamp loading complex is loaded onto DNA. The DNA Pol δ and/or e complex extends the leading strand and Pol α synthesizes the lagging strand. Claspin is bound to the fork and Chk1 has a role in the maintenance of fork stability.
(3) Mediators and Chk kinases 1 and 2 are recruited to sites of damage and are phosphorylated within nuclear foci. While mediators can contribute to the effectiveness of the S phase checkpoint in an effector kinase-independent manner, activated Chks are released from foci and phosphorylate their targets CDC25 and perhaps p53. p53 transcriptional activity might be modulated to ensure the reversibility of the arrest.
Studies have shown that HuCdc6 can trigger a checkpoint response involving Chk1 as a result of mitotic catastrophe or the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition, however how Cdc6 itself mediates cell cycle has not yet been known.
4.3 Cdc6 inhibits DNA replication without activating ATM/ATR-dependent checkpoint pathways
4.3.1 Immunoblot analysis
Previous works show that presence of Cdc6 during S phase is essential for a checkpoint kinase, Chk1, to become activated in response to replication inhibition (Oehlmann, 2004). During mitotic exit in Saccharomyces cerevisiae, Cdc6 cooperates with Sic1 to directly inactivate Cdks (Calzada et al., 2001), which is thought to be mediated by activation of the Chk1 checkpoint kinase. Therefore, I set out to examine whether inhibition of DNA replication by Cdc6 observed in this study involved ATM/ATR-dependent checkpoint pathways.
Figure 1-4-3. Cdc6 addition did not activate checkpoint pathways. Nuclei were isolated
from extracts treated with aphidicolin or GST-Cdc6 (700 nM) in the presence or absence of caffeine (5 mM) and immunoblotted for phospho-Chk1.
After an incubation of sperm nuclei in Cdc6-supplemented extracts for 90 minutes, nuclei were isolated and immunoblotted for phospho-Chk1-S345, as Chk1 is phosphorylated on Ser345 by ATR in response to a variety of genomic insults (Liu et al., 2000; Guo et al., 2000; Zhao and Piwnica-Worms, 2001). Aphidicolin is known to
caffeine
Chk1-P
H3
trigger ATR-dependent checkpoint pathways resulting in the phosphorylation of Chk1 and was used as a control. Caffeine is used in biochemical experiments as an inhibitor of ATM and ATR.
Figure 1-4-3 shows that aphidicolin induced Chk1 phosphorylation, which was blocked by addition of caffeine consistent with it being a consequence of activation of ATM-ATR checkpoint pathways. On the other hand, no phospho-Chk1 band was detected in Cdc6-supplemented extracts. This result suggested the possibility to exclude the activation of ATM/ATR-dependent checkpoints from potential mechanisms that might have involvement in Cdc6-dependent replication inhibition.
4.3.2 Replication assays by the incorporation of radioactivity
To further assess the involvement of caffeine-sensitive checkpoint pathways, I adopted replication assay in the presence of GST-Cdc6 to see whether its inhibition was blocked by addition of caffeine.
Figure 1-4-4. Cdc6 did not activate caffeine-sensitive checkpoint pathways. Extracts
supplemented with buffer or caffeine were treated with either EcoRI or GST-Cdc6 (700 nM). DNA synthesis was measured after 90 min of incubation.
Sperm chromatin was added to extracts supplemented with EcoRI or GST-Cdc6 in the presence or absence of caffeine and the amount of synthesized DNA was measured after an incubation of 90 min. Consistent with previous results, EcoRI blocked DNA replication, which was rescued by addition of caffeine, reflecting the inhibition of EcoRI-dependent Chk2 activation by caffeine. However, addition of caffeine did not overcome the inhibition of DNA replication caused by the addition of GST-Cdc6 (Figure 1-4-4). Therefore, I speculate that inhibition of DNA replication by Cdc6 in undisturbed S phases does not involve ATM/ATR-dependent checkpoint pathways.
5 Effect of addition of Cdc6 during the course of DNA
replication.
Next, I sought to determine the step at which DNA replication was arrested by Cdc6 addition. To address the issue, I added GST-Cdc6 at different time points during DNA replication and measured DNA synthesis by the radioactivity incorporated into DNA (Figure 1-5-1).
Figure 1-5-1. Cdc6 inhibited DNA replication at an earlier step. A scheme of the assay (left).
GST-Cdc6 (700 nM) was added at indicated times after sperm addition and DNA synthesis was measured after 90 min of incubation in total (right).
Whereas the addition of GST-Cdc6 at an earlier stage caused complete inhibition of DNA synthesis, adding GST-Cdc6 later than 30 min did not show any repression. In our experimental system, pre-RC formation reaches peak around 15-20 min while origin firing occurs followed by DNA synthesis during the next 60-70 min. Therefore, I decided to clarify the influence of excess Cdc6 on licensing, origin firing or elongation.
extracts
sperm DNA
measurement of DNA synthesis
90 min after the addition of sperm DNA
rCdc6 at indicated times
[a-32
6 Effect of Cdc6 on DNA synthesis
6.1 DNA Polymerases
DNA polymerase proceeds along a single-stranded molecule of DNA, recruiting free deoxy-nucleotide-triphosphates (dNTPs) to create hydrogen bond with their appropriate complementary nucleotide on the template strand (A with T and G with C), and to form a covalent phosphodiester bond with the previous nucleotide of the synthesizing strand. The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand. There are different forms of DNA polymerases, e.g. pol a, d, e, z etc. Since DNA polymerase cannot start synthesizing de novo on a bare single strand, it needs a primer with a 3'OH group onto which it can attach a dNMP.
6.2 Aphidicolin: a DNA polymerase inhibitor
Aphidicolin, a tetracyclic diterpenoid isolated from Ceph-alosporium aphidicola is a potent inhibitor of DNA replication. It specifically inhibits replicative DNA polymerases, and was found to inhibit mitotic division of sea urchin embryos while not affecting nondividing cells. The drug proved to be instrumental in identifying pol a as a major eukaryotic replicative polymerase. Aphidicolin also specifically inhibits pol d and e, while not affecting DNA methylation or RNA, protein, and nucleotide biosynthesis.
Aphidicolin competes with each of the four dNTPs or binding to a pol a-DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol a-DNA-aphidicolin ternary complex. This decreases the size of nascent strands and the inhibition of replication
forks in turn activates an ATM-ATR checkpoint response that inhibits further origin firing.
6.3 Strategy and assay designing
The data described in the previous chapters show that Cdc6 is likely to regulate DNA replication at an early stage of DNA replication. The final step in replication initiation is the loading of the replicative DNA polymerases. After origin unwinding, pola is recruited to origins and synthesizes short RNA primers for leading and lagging strand synthesis. As a step to find the outer boundary of the window of action of additive Cdc6, its effect on DNA polymerase was examined next. To pursue this, an experiment was designed in light of chromatin transfer technique in previous reports.
In this experiment, aphidicolin, an inhibitor of DNA polymerase, was added to extracts and incubated for 60 min at 23˚C. In these extracts DNA replication stops before DNA synthesis starts. The nuclei were then isolated (aphidicolin nuclei) to wash aphidicolin away and incubated for a further 90 min in extracts supplemented with buffer, rCdc6 or aphidicolin. In these extracts the stalled replication fork regains its ability to start DNA synthesis. If an excess of Cdc6 has a negative effect on an elongation of the nascent strand, DNA replication will be impaired in the extracts containing excess Cdc6.
6.4 DNA synthesis was not inhibited by additive Cdc6
As was mentioned above, aphidicolin blocks DNA polymerases, therefore, chromatin preincubated with aphidicolin arrests before elongation. Nuclei were then isolated in nuclear isolation buffer and transferred to fresh extracts supplemented with buffer, GST-Cdc6 or aphidicolin.
Figure 1-6-1. Cdc6 did not affect nascent DNA synthesis. A schematic representation of the
nuclear transfer assay (left). Amount of DNA synthesized in extracts containing buffer, aphidicolin (40 ng/mL) or GST-Cdc6 (700 nM) supplemented with untreated sperm chromatin or aphidicolin nuclei (right).
Figure 1-6-1 shows that aphidicolin nuclei fully supported DNA replication even in the presence of rCdc6 while non-treated sperm nuclei failed to replicate in the presence of additive Cdc6. This result indicates that Cdc6 inhibits DNA replication at a step that is before the synthesis of nascent DNA.
extracts
23°C 60 min
isolation of aphidicolin nuclei (arrested before DNA synthesis starts)
aphidicolin
+
extract +/- rCdc6 measurement of DNA synthesis 23°C 90 min sperm DNA7 Effect of additive Cdc6 on Cdk2-dependent steps
Now the targets are concentrated on to a window covering a range from licensing to origin firing. If we look at this stage, we can see two kinases acting independently and parallelly – Cdc7/Dbf4 (DDK) and Cdk2. First Cdc7 is loaded onto chromatin only after origins become licensed and phosphorylates mainly Mcm2, Mcm4 and Mcm7. This phosphorylation further stimulates activation of Cdk2, and depending on Cdk2 function, GINS complex and Cdc45 are recruited onto chromatin and activate the putative Mcm2-7 helicase. As it has been shown in previous chapter that Cdc6 inhibits DNA replication at a step before DNA synthesis, the next interest was to investigate the effect on Cdk2.
7.1 CDKs: the major regulator of the cell cycle
CDKs are proline-directed serine/threonine protein kinases that play essential roles in the regulation of eukaryotic cell division. The enzymatic activity of CDKs is modulated by protein-protein interactions as well as by both inhibitory and activating phosphorylations. Association with regulatory subunits named cyclins that are synthesized and degraded in a cell-cycle-dependent manner activates CDKs. Cyclin binding provides the CDK with targeting domains important for substrate selection and subcellular localization, which in turn determine the biological specificity.
The first CDK to be identified, Cdc2, was initially discovered as a gene essential for both G1/S and G2/M transitions in the S. pombe cell cycle. Cdc2 homologues were subsequently found in all eukayotes, including humans, where it is referred to as Cdk1.
involved in cell division. Cyclins comprise a diverse family of proteins that were identified in sea urchin eggs and later found to be present in all organisms from yeast to man. They all share a conserved sequence of 100 amino acids, named the cyclin box, which is necessary for CDK binding and activation.
A broadly accepted view of the cell cycle considers that cyclin D-bound Cdk4 and Cdk6 are involved in the early G1 phase, whereas Cdk2 bound to either cyclin E or cyclin A regulates the G1/S transition and S phase progression, respectively. Further cell cycle progression is regulated by Cdk1-cylin A at the S/G2 transition and Cdk1-cyclin B at the G2/M transition and M phase progression. Recently, the generation of gene-targeted mice has called into question the importance of Cdk4 and Cdk6 for cell cycle entry after mitogenic stimuli as well as the requirement of Cdk2 for the mitotic cell cycle. Likewise, proliferation of mouse cells appears to be much less dependent on D- and E-type cyclins than was originally anticipated. These findings have led to the proposal of a revised model of the mammalian cell cyle.
7.2 p21: an inhibitor of CDK
p21 protein regulate cyclin–CDK complexes in the cytoplasm. p21 acts as a bridge between cyclin D and Cdk4 to promote their association. Following binding, p21 protein enhances the nuclear translocation of the complex. Once in the nucleus, nascent cyclin-D–Cdk4 complexes titrate p21 from Cdk2, thereby inducing cell-cycle progression. Upon cell-cycle arrest, the level of p21 proteins increases, saturate D-type proteins and then bind to cyclin-E–Cdk2 to block the catalytic activity of the kinase. Based on structural studies, it is believed that an α-helix of p21 protein initiates a first contact with the cyclin, and that a second helix then inserts deep inside the catalytic cleft of the CDK subunit, thereby blocking ATP loading. Important Cdk2
conformational changes further lock the catalytic cleft in an inactive form. Surprisingly, the vast majority of cyclin-D̽Cdk4 complexes contain p21 or p27, predicting that cells should contain no active Cdk4. Nevertheless, several studies argue that this conformation represents the active state of the cyclin D complex and that low concentrations of p21 protein inhibit Cdk2 but not Cdk4/6. Since the activated form of Cdk2 is located in the nucleus, only nuclear forms of p21 protein should probably be considered as catalytic inhibitors of Cdk2.
7.3 Strategy and assay designing
I exploited similar protocol described in chapter 6 using Cdk2 inhibitor, p21, instead of aphidicolin in order to determine whether Cdc6 influences Cdk2-dependent processes (Strausfeld et al., 1994; Chong et al. 1995; Mimura and Takisawa 1998). As it was described in previous section, p21 inhibits Cdk2 thus blocking Cdc45 loading and the initiation of DNA replication but not licensing. Also blocking Cdk2 activity by addition of p21 has no significant effect on the activity of Cdc7/Dbf4 kinase. Therefore, in p21 treated extracts, DNA replication stops after Cdc7-dependent steps but before Cdc45 dependent unwinding of DNA. So, if the chromatin isolated from p21 treated extracts fail to replicate in extracts containing excess Cdc6, it can be said that Cdc6 has a negative effect on Cdk2-dependent steps.
7.4 DNA replication is inhibited at a step before Cdk2-dependent pathways
Figure 1-7-1. Cdc6 did not inhibit Cdk2-dependent pathways. A scheme of the nuclear
transfer experiment (left). Amount of DNA synthesized in extracts containing buffer, p21 (5 ng/mL) or GST-Cdc6 (700 nM) supplemented with untreated sperm chromatin or p21 nuclei (right).
Nuclei were isolated from extracts treated with p21 (p21 nuclei) to arrest chromatin before Cdk2 activation. Similar to aphidicolin nuclei, p21 nuclei were also fully competent to support DNA replication in rCdc6-supplemented extracts (Figure 1-7-1). These results suggest that Cdc6 inhibits DNA replication at a step earlier than Cdk2-dependent events.
extracts
23°C 45 min
isolation of p21 nuclei (arrested before origin firing)
p21
+
extract +/- rCdc6 measurement of DNA synthesis 23°C 90 min sperm DNA8 Influence of additive Cdc6 on origin licensing
8.1 Introduction
8.1.1 ORC
Pre-RC assembly at origins of DNA replication is essential to license chromosomes before initiation of DNA synthesis occurs during S phase of the cell division cycle. Formation of pre-RC requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ORC, a six-subunit, ATP-dependent DNA-binding protein, binds to specific DNA sequences at origins of DNA replication and is the foundation for pre-RC assembly. The Orc1 and Orc5 subunits are known to interact with ATP, but only the interaction between the Orc1 subunit and ATP is required for DNA binding and is essential in yeast. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Once ORC is bound to origin DNA, the ORC ATPase is reduced or blocked. Binding of Cdc6 to ORC is a key step in the assembly of the pre-RC.
8.1.2 Cdt1 and Geminin
Cdt1 is essential in G1 for licensing of origins for DNA replication and is inhibited in S phase both by binding to geminin and degradation by proteasomes. Overexpression of Cdt1 or downregulation of geminin causes re-replication of DNA. DNA re-replication causes genomic instability, activation of checkpoint pathways and eventually cell death or cell transformation. Therefore, Cdt1 is tightly regulated during the cell cycle to make sure that cells do not re-replicate their DNA. Phosphorylation of Cdt1 by Cdk2
promotes its binding to Skp2-SCF E3 ubiquitin ligase, but the Cdk2/Skp2 mediated pathway is not essential for the degradation of Cdt1 in S phase.
7.1.3 Mcm (Mini chromosome maintenance) proteins
The Mcm2-7 complex controls the once per cell cycle DNA replication in eukaryotic cells. In a process known as DNA replication licensing, it primes chromatin for DNA replication by binding origins of DNA replication during the late M to early G1 phase of the cell cycle. Activated by S phase promoting protein kinases, the origin-bound Mcm2-7 complexes unwind the double stranded DNA at the origins, recruit DNA polymerases and initiate DNA synthesis. Coupled with the initiation of DNA replication in the S phase, the Mcm2-7 complexes move away from replication origins as a component of the DNA replication fork, likely serving as DNA helicases. Their departure deprives replication origins the ability to re-initiate DNA replication for the reminder of the cell cycle. Because of its vital role in genome duplication in proliferating cells, deregulation of the Mcm2-7 function results in chromosomal defects that may contribute to tumorigenesis.
The Mcm proteins are highly expressed in malignant human cancer cells and pre-cancerous cells undergoing malignant transformation. They are not expressed in differentiated somatic cells that have been withdrawn from the cell cycle. Therefore, these proteins are ideal diagnostic markers for cancer and promising targets for anti-cancer drug development.
8.2 Cdc6 does not affect functional pre-RC formation
8.2.1 Immunoblot analysis
Next, I examined the chromatin binding of various proteins during pre-RC formation. Chromatin was isolated from egg extracts supplemented with or without rCdc6. Consistent with a previous paper (Coleman et al., 1996), Orc1, Cdc6 and Cdt1 associated rapidly with chromatin, and loading of Mcm2-7 followed (Figure 1-8-1). Importantly, no significant alteration in the chromatin binding of Orc1, Cdt1 and Mcm2-7 was observed in rCdc6-supplemented extracts although there was slight decrease in Mcm2, 4 and 6 loading. I later confirmed that this decrease occurred at concentrations (800 nM) of Cdc6 higher than that needed to cause sufficient inhibition (700 nM), and I also checked that even this lower amount of Mcm2-7 complex is enough to trigger DNA replication in fresh extracts (data not shown). Therefore, at present stage, I would prefer to say that rCdc6 does not significantly alter chromatin binding of Mcm2-7 proteins to inhibit DNA replication.
Figure 1-8-1. Chromatin binding of proteins during origin licensing. Chromatin was
isolated at indicated time points from extracts supplemented with buffer or rCdc6 (800 nM). Isolated chromatin was subjected to SDS-PAGE and immunoblotting.
Previous findings show that geminin binds Cdt1 and inhibits Mcm2-7 loading resulting in stabilization of ORC and Cdc6 on chromatin at this condition. Consistent with this, in the presence of geminin, Cdc6 remained at high levels on the chromatin and Mcm2-7 loading was completely inhibited. These results suggest that Mcm2-7 loaded in rCdc6-supplemented extracts are functional for the licensing and presence of excess Cdc6 causes no significant alteration in the chromatin binding of ORC, Cdt1 or Mcm2-7.
8.2.2 Additive Cdc6 did not hamper functional pre-RC formation
I next attempted to confirm that the Mcm2-7 proteins loaded in rCdc6-supplemented extracts are competent for DNA replication. Sperm DNA was incubated in egg extracts supplemented with buffer or rCdc6 and chromatin was isolated after an incubation for 30 min to allow sufficient time for the Mcm2-7 loading.
Figure 1-8-2. Mcm2-7 loaded in Cdc6-supplemented extracts are fully functional. A
scheme of the chromatin transfer experiment (left). Sperm chromatin was incubated in extracts supplemented with buffer or rCdc6 for 30 min. Chromatin was isolated and transferred to fresh
extracts+/- rCdc6 23°C 30 min measurement of DNA synthesis 23°C 90 min sperm DNA fresh extract + geminin chromatin isolation
extract containing geminin. DNA synthesis was measured after 90 min in the 2nd incubation (right).
Chromatin was then isolated and transferred to fresh geminin-treated extracts so that the origins licensed in the first extract could only initiate replication in the second extract. As the result, incorporation of radioactivity confirmed that DNA replication activity was almost the same for chromatin isolated from rCdc6-supplemented extracts to that from non-treated extracts (Figure 1-8-2), suggesting that addition of Cdc6 does not affect functional origin licensing and the Mcm2-7 complexes can support DNA replication sufficiently.
I further investigated whether Cdc6 destabilized chromatin-bound licensing factors by supplementing rCdc6 after sperm addition. To achieve this, rCdc6 was added at indicated timepoints (Figure 1-8-3) after the addition of sperm chromatin. Chromatin was isolated after a total incubation of 20 min.
Figure 1-8-3. rCdc6 did not destabilize Cdt1 or Mcm2-7. rCdc6 was added at indicated
timepoints after the addition of sperm chromatin. Chromatin was isolated after a total incubation of 20 min and immunoblotted subsequently.
chromatin binding of Cdt1. However, the amount of Mcm2-7 loaded onto chromatin increased compared to when Cdc6 was added simultaneously with sperm chromatin (0 min). This result suggests that Cdc6 does not at least destabilize chromatin-bound Cdt1 or Mcm2-7. The precise mechanism of the increase in Mcm2-7 loading is not clear yet.
Next, I sought to determine whether inhibition of DNA replication was due to Cdc6-Cdt1 imbalance. Although Cdt1 can bind chromatin independently of Cdc6, a strict sequential binding of Cdc6 and Cdt1 is required for functional origin licensing (Tsuyama et al., 2005) and apparently proper interaction between Cdc6 and Cdt1 is necessary for functional pre-RC formation. Therefore, it is possible that excess amount of Cdc6 may hamper this interaction thereby blocking DNA replication. To test this possiblity, we added various amount of GST-Cdt1 in the presence or absence of caffeine to rCdc6-supplemented extracts and DNA synthesis was measured subsequently.
Figure 1-8-4. Inhibition of DNA replication by Cdc6 was not rescued by addition of Cdt1.
Indicated amounts of GST-Cdt1 was added to extracts supplemented with rCdc6 plus or minus caffeine (5 mM). DNA synthesis was measured 90 min after the addition of sperm DNA.
Caffeine was added to exclude the influence of ATM/ATR-dependent checkpoint pathways because it has been shown in previous studies that excess Cdt1 can induce a checkpoint response (Davidson et al., 2006). As the result shows, addition of GST-Cdt1 could not rescue the inhibition of DNA replication caused by rCdc6 addition suggesting strongly that this blockage of DNA replication was not due to the imbalance of Cdc6-Cdt1 amount.
